(WHS-P27) ROLE OF HYALURONAN TOPOGRAPHY ON EPITHELIAL TO MESENCHYMAL TRANSITION OF KERATINOCYTES
Friday, May 17, 2024
7:30 AM – 5:00 PM East Coast USA Time
Introduction: Epithelial-to-mesenchymal transition (EMT) is a well studied biological phenomenon that takes place during various biological processes including wound healing, Type II EMT. Embryos undergo scarless wound healing while adult wound healing creates a scar. The wound healing extracellular matrix (ECM) varies between adults and embryos. Embryos have higher levels of hyaluronan (HA) and type III collagen fibers, while the adult ECM is marked by denser, aligned collagen bundles and reduced HA. Various studies have reported that HA helps keratinocyte proliferation and migration during wound healing. It remains unknown how different HA topographies affect the proliferation and migration of keratinocytes. To address this gap, we created synthetic matrix analogs that are capable of representing different HA topographies using hyaluronan binding peptide (HABP) studied the role of HA topography on EMT.
Methods: Synthesis and Surface Modification of PCL Fibers. Polycaprolactone (PCL) fibers with diameters 0.5µm and 5µm were made through electrospinning and functionalized with HABP through aminolysis as previously reported [4]. Fiber characterization was performed through SEM and water contact angle (CA) analysis. Cell Proliferation and Migration. Primary human epidermal keratinocytes were used to study EMT. Cell proliferation was studied using Ki67, pico green, and presto blue assays. Cell migration was studied using a transwell migration assay. EMT Marker Quantification. The concentration of common EMT markers was found using western blot. Cell Morphology. Cell morphology was analyzed through fluorescent microscopy where the nucleus and f-actin were stained. In the epithelial state, cells display cortical actin in the membrane cortex, which transitions into stress fibers in the mesenchymal state. Results We found PCL scaffolds had an average CA of 82.84 ± 17.48° and with the addition of HA the average CA was 77.57 ± 16.52°. With just the addition of HABP to the scaffolds the average CA was 11.88 ± 19.35° and with the addition of HABP and HA the average CA was 4.38 ± 10.72°. The addition of the peptides makes the surface hydrophilic with HABP making the surface completely hydrophilic. Ongoing experiments are currently focusing on understanding the role that HA topography plays has on EMT by measuring the concentration EMT markers. Fluorescent images of epithelial cells on the different surfaces will be taken to determine if the addition of a peptide causes cells to take on more of a mesenchymal shape. The results of this experiment will shed light on how topography influences the factors that trigger cells to undergo EMT. The goal of this study was to determine the effect of different the impact of various hyaluronan topographies on cells during Type II EMT.